Gap Junctions in the Gap Junctions in the SCNSCN

YiDing YuYiDing Yu

MCB 186 Circadian BiologyMCB 186 Circadian Biology

Autonomous OscillatorsAutonomous Oscillators

Single-cell autonomous Single-cell autonomous oscillators in the SCN oscillators in the SCN (Welsh 1995)(Welsh 1995)

Synchronized firing in vivoSynchronized firing in vivo Heterogeneity within the Heterogeneity within the

SCNSCN rhythmicity (phase, period)rhythmicity (phase, period) neurotransmitterneurotransmitter environmental responseenvironmental response rhythms controlledrhythms controlled

Multiple PhenotypesMultiple PhenotypesRostral Caudal (A E) Moore et al. 2002

How are single-cell oscillators How are single-cell oscillators synchronized in vivo?synchronized in vivo?

Gap JunctionsGap Junctions NeurotransmittersNeurotransmitters

GABAGABA Vasoactive intestinal Vasoactive intestinal

polypeptide (VIP)polypeptide (VIP) Gastrin-releasing peptide Gastrin-releasing peptide

(GRP)(GRP) Prokineticin 2 (PK2)Prokineticin 2 (PK2)

Maintain Rhythmicity?Maintain Rhythmicity?

Aton et al. 2005Aton et al. 2005

Neuronal Gap JunctionsNeuronal Gap Junctions Spike-for-spike Spike-for-spike

synchronizationsynchronization Connexins expressed in the Connexins expressed in the

SCNSCN Connexin-36 found in Connexin-36 found in

murine and human neuronal murine and human neuronal gap junctionsgap junctions

Long et al. 2005Long et al. 2005 Electrical coupling between Electrical coupling between

25% of neighboring cells25% of neighboring cells Electrical coupling lost in Electrical coupling lost in

connexin-36 (Cx36) KO miceconnexin-36 (Cx36) KO mice

Soehl et al. 2005

HypothesisHypothesis

AA. Gap junctions are not necessary to maintain . Gap junctions are not necessary to maintain rhythmicity of individual SCN cellsrhythmicity of individual SCN cells

BB. Gap junctions are integral to maintaining synchrony . Gap junctions are integral to maintaining synchrony between coupled cellsbetween coupled cells

Are gap junctions necessary for maintaining Are gap junctions necessary for maintaining rhythmicity among SCN cells?rhythmicity among SCN cells?

To what extent do gap junctions maintain synchrony To what extent do gap junctions maintain synchrony between coupled cells?between coupled cells?

Protocol (in vitro)Protocol (in vitro)Experiment 1:Experiment 1:Rhythmicity in WT cellsRhythmicity in WT cells Electrode recording in Electrode recording in

various regionsvarious regions Dense vs. sparse neuronal Dense vs. sparse neuronal

populationpopulation Dorsal shell vs. ventral coreDorsal shell vs. ventral core Neurotransmitter output Neurotransmitter output

(AVP, VIP, GRP)(AVP, VIP, GRP)

Experiment 2:Experiment 2:Synchronization in WT cellsSynchronization in WT cells Electrode recordings to find Electrode recordings to find

coupled SCN neuronscoupled SCN neurons Apply transmitter receptor Apply transmitter receptor

antagonists to eliminate fast antagonists to eliminate fast chemical synaptic chemical synaptic transmissiontransmission

APPLY TREATMENT:APPLY TREATMENT: Quinine (Cx36 specific) Quinine (Cx36 specific)TREATMENT WASHOUT TREATMENT WASHOUT Restore WT phenotype Restore WT phenotypePositive control:Positive control: Cx36 KO mice Cx36 KO miceNegative control: Negative control: Untreated WT miceUntreated WT mice

Predicted ResultsPredicted Results

Experiment 1: Experiment 1: RhythmicityRhythmicity No effect on individual No effect on individual

cell rhythmicity upon cell rhythmicity upon treatment or washout, treatment or washout, regardless of SCN regionregardless of SCN region

Experiment 2: SynchronizationExperiment 2: Synchronization Gap junction blocker treatment: Loss of synchrony Gap junction blocker treatment: Loss of synchrony

between coupled cells; similar to Cx36 KObetween coupled cells; similar to Cx36 KO Washout: Restore WT synchronyWashout: Restore WT synchrony

Long et al. 2005

Implications & Further StudiesImplications & Further Studies

All cells remain rhythmic in experiment 1:All cells remain rhythmic in experiment 1: Gap junctions do not function to maintain Gap junctions do not function to maintain rhythmicity of oscillator cellsrhythmicity of oscillator cells If some cells become arrhythmic: If some cells become arrhythmic: Gap junctions Gap junctions

may play a role in the regulation of some SCN may play a role in the regulation of some SCN cells by other autonomous cell oscillators cells by other autonomous cell oscillators Is Is this prevalent in a particular region of the this prevalent in a particular region of the SCN? SCN?

Implications & Further StudiesImplications & Further Studies

Coupled WT cells lose synchrony upon treatment Coupled WT cells lose synchrony upon treatment (experiment 2): (experiment 2): Cx36 gap junctions are integral in Cx36 gap junctions are integral in maintaining coupling maintaining coupling Does coupling occur Does coupling occur predominately in certain areas of the SCN?predominately in certain areas of the SCN? If cells remain synchronized:If cells remain synchronized: Cx36 gap junctions are not Cx36 gap junctions are not

necessary in maintaining coupling; Cx36 KO phenotype necessary in maintaining coupling; Cx36 KO phenotype confounded confounded

Possible other connexins involved? (Octanol, halothane)Possible other connexins involved? (Octanol, halothane) Effects of gap junctions between neurons and glia? Glia and Effects of gap junctions between neurons and glia? Glia and

glia?glia?